Compositions and methods for enhanced tumor cell immunity in vivo

ABSTRACT

The invention provides a method of preventing or reducing the severity of a cancer in a subject by stimulating the subject&#39;s immune response against the cancer. The invention provides, for example, a method of stimulating an immune response in a subject by administering to the subject tumor cells that are substantially similar to the subject&#39;s cancer cells and that are genetically modified to reduce or inhibit the expression of one or more immunosuppressive agents. The invention also provides a method of preventing or reducing the severity of cancer in a subject by stimulating the subject&#39;s immune response against the cancer by administering to the subject tumor cells that are substantially similar to the subject&#39;s cancer cells and that are genetically modified to prevent the expression of an immunosuppressive agents and, in combination with the genetically modified tumor cells, an immunostimulatory agent. The invention further provides compositions useful for practicing the claimed methods.

This application is a continuation, of application Ser. No. 08/968,986,filed Nov. 12, 1997, now U.S. Pat. No. 6,120,763, issued Sep. 19, 2000,which is a continuation of application Ser. No. 08/276,694, filed Jul.18, 1994, now U.S. Pat. No. 5,772,995, issued Jun. 30, 1998.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to anti-tumor vaccines and, moreparticularly, to the use of gene therapy to augment immunologicalfunctions in response to anti-tumor vaccines.

2. Background Information

Recent advances in our understanding of the biology of the immune systemhave lead to the identification of cytokines as important modulators ofimmune responses. Cytokines produced by lymphocytes are termedlymphokines. These agents mediate many of the immune responses totumors. Several cytokines have been produced by recombinant DNAmethodology and evaluated for their anti-tumor effects.

The administration of lymphokines and related immunomodulators hasproduced some positive responses in patients with various types ofneoplasms. However, current cytokine administration is frequentlyassociated with toxic effects that limit the therapeutic value of theseagents. For example, interleukin-2 (IL-2) is an important lymphokine inthe generation of anti-tumor immunity. In response to tumor antigens, asubset of lymphocytes, helper T cells, secrete a small amount of IL-2,which acts locally at the site of tumor antigen stimulation to activatecytotoxic T cells and natural killer cells that mediate systemic tumorcell destruction.

In addition to immunostimulatory agents such as cytokines, whichpositively regulate immune effector functions, there also existmolecules that exhibit immunosuppressive activity. Theseimmunosuppressive agents, if aberrantly regulated, can have detrimentaleffects on the induction of systemic immunity. For example, one or moreisoforms of transforming growth factor-β (TGFβ) can be animmunosuppressive agent that is secreted by many tumor types. Culturesupernatants from tumor cells that secrete an immunosuppressive form ofTGFβ can reduce tumor specific cytotoxicity in vitro. In these in vitrocytotoxicity assays, inhibition of TGFβ activity by expression ofantisense TGFβ can enhance tremor cell cytotoxicity.

Due, in part, to endogenous concentrations of immunosuppressive agentsin a subject having a cancer, it cannot be predicted whether inhibitingthe secretion of an immunosuppressive agent by the tumor cell willrender the tumor cell immunogenic in vivo. For example, TGFβ secreted bycancer cells can circulate throughout a cancer patient and generallyimmunocompromise the patient. As a result, administration of tumor cellsthat are substantially similar to the patient's cancer cells and thatare genetically modified to prevent the expression of animmunosuppressive agent would not necessarily be expected to stimulatethe patients immune response against the cancer cells. Similarly, theresults of in vitro models of anti-tumor immune responses do notreliably predict the outcome of related immune system manipulations invivo.

The modulation of cytokine concentrations has been attempted as a meansto enhance a cancer patient's immune response toward target cancercells. For example, intravenous, intralymphatic or intralesionaladministration of IL-2 has produced clinically significant responses insome cancer patients. However, severe toxic effects such as hypotensionand edema limit the dose and efficacy of intravenous and intralymphaticIL-2 administration. The toxicity of systemically administeredlymphokines is not surprising as these agents mediate local cellularinteractions and normally are secreted only in very small quantities. Inaddition, intralesional administration of IL-2 can be difficult toaccomplish and can cause significant patient morbidity.

To circumvent the toxicity of systemic cytokine administration, analternative approach involving cytokine gene transfer into tumor cellshas produced anti-tumor immune responses in several animal tumor models.in these studies, the expression of cytokines following cytokine genetransfer into tumor cells resulted in a reduction in tumorigenicity ofthe cytokine-secreting tumor cells when implanted into syngeneic hosts.Reduction in tumorigenicity occurred using IL-2, gamma-interferon orinterleukin-4. In studies employing IL-2 gene transfer, the treatedanimals also developed systemic anti-tumor immunity and were protectedagainst subsequent tumor cell challenges with unmodified parental tumorcells. Similar inhibition of tumor growth and protective immunity alsowas demonstrated when immunizations were performed with a mixture ofunmodified parental tumor cells and tumor cells that were geneticallymodified to express IL-2. No toxicity was associated with localizedlymphokine transgene expression in these animal studies.

Cytokines also have been expressed In heterologous cell types such asfibroblasts, which were coinjected into a cancer patient with thepatient's own cancer cells. Coinjection of cytokine-expressing cellsyielded a similar induction of systemic anti-tumor immunity as producedby cytokine-expressing tumor cells. Nevertheless, while these genetransfer procedures can provide significant anti-tumor immunity comparedto other methods, a significant fraction of the patients do not respondoptimally to such therapy. Thus, there exists a need to provide moreeffective methods to prevent or reduce the severity of a cancer in apatient. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of preventing or reducing theseverity of a cancer In a subject by stimulating the subject's immuneresponse against the cancer. For example, a cancer patient can beimmunized with tumor cells that are substantially similar to thesubject's cancer cells but that are genetically modified to prevent theexpression of at least one immunosuppressive agent.

The invention also provides a method of stimulating a subject's immuneresponse against a cancer by administering tumor cells that aresubstantially similar to the subject's cancer cells but that aregenetically modified to prevent the expression of at least oneimmunosuppressive agent and to express at least one immunostimulatoryagent such as a cytokine or a known tumor antigen. The invention furtherprovides a method of stimulating a cancer patient's immune responseagainst the cancer by co-administering to the subject animmunostimulatory agent such as an adjuvant, a cytokine orcytokine-expressing cells (CE cells), which are cells that aregenetically modified to express a cytokine, and tumor cells that aresubstantially similar to the subject's cancer cells and that aregenetically modified to prevent the expression of at least oneimmunosuppressive agent.

The invention also provides compositions useful for preventing orreducing the severity of a cancer in a subject by stimulating thesubject's immune response against the cancer. A composition of theinvention can contain tumor cells that are substantially similar to thesubject's cancer cells and that are genetically modified to prevent theexpression of at least one immunosuppressive agent. In addition, ifdesired, the genetically modified tumor cells can be further modified toexpress an immunostimulatory agent such as a cytokine or a known tumorantigen. A composition of the invention also can contain animmunostimulatory agent such as an adjuvant, a cytokine or CE cells andtumor cells that are substantially similar to the subject's cancer cellsand that are genetically modified to prevent the expression of at leastone immunosuppressive agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1.A. and 1.B. illustrate the in vitro anti-tumor lytic activity oflymph node effector cells from rats immunized with unmodified 9Lgliosarcoma cells (FIG. 1.A.; 9L Immunized) or with 9L cells modified toexpress and secrete IL-2 (FIG. 1.B.; 9L/LNCX/IL-2 immunized). Lymph nodecells were harvested from immunized animals and stimulated in vitro inthe presence of 50 BRMP units IL-2/ml medium with either unmodified 9Lcells or 9L cells that were genetically modified to express an antisenseTGFβ. Target cells consisted of unmodified 9L cells.

FIG. 2 illustrates the effect of immunization on the survival of miceinjected with murine ovarian teratoma (MOT) tumor cells. Mice wereimmunized with either unmodified MOT cells (MOT), MOT cells geneticallymodified to express antisense TGFβ (MOT/TGFβas), unmodified MOT cellsand CE cells (MOT+IL2) or a combination of MOT cells geneticallymodified to express antisense TGFβ and CE cells (MOT/TGFβas+IL-2).Control mice were not immunized (Unimmunized). Numbers on the rightindicate surviving mice/total mice tested.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions that can effectively preventor reduce the severity of a cancer in a subject and methods for usingthe compositions. In particular, the invention provides compositionscontaining tumor cells that are genetically modified to reduce orinhibit the expression of one or more immunosuppressive factors normallyproduced by the tumor cells. Tumor cells that produce immunosuppressivefactors are known in the art and are present, for example, incarcinomas, sarcomas, gliomas, melanomas, lymphomas and leukemias(Sulitzeanu, Adv. Canc. Res. 60:247-267 (1993), which is incorporatedherein by reference). Whether a cancer is producing an immunosuppressiveagent can be readily determined using methods as described herein orotherwise known in the art.

A composition of the invention contains tumor cells that are geneticallymodified to provide an enhanced systemic immune response against acancer by preventing or reducing the expression of one or moreimmunosuppressive agents by the tumor cells. It is recognized,therefore, that prior to genetic modification, the tumor cells arecharacterized, in part, by their ability to express an immunosuppressiveagent.

A composition of the invention can contain the genetically modifiedtumor cells, alone, or in combination with an immunostimulatory agentsuch as a cytokine or cytokine-expressing cells (CE cells), which arecells that are genetically modified to express a cytokine. A compositionof the invention also can contain tumor cells that are geneticallymodified to reduce or inhibit the expression of an immunosuppressiveagent and are further genetically modified to express animmunostimulatory agent such as a cytokine or a known tumor antigen. Thecompositions of the invention are advantageous over existing cancervaccines because the disclosed compositions, when administered to asubject, can provide superior modulation of the subject's immunefunctions.

As used herein, the term “preventing or reducing the severity of acancer” means that the clinical signs or symptoms of the cancer in asubject are indicative of a beneficial effect to the subject due tostimulation of the subject's immune response against the cancer. Theprevention of a cancer can be indicated by an increased time before theappearance of a cancer in a subject that is predisposed to developing acancer. A subject can be predisposed to developing a cancer due, forexample, to a genetic predisposition or to exposure to a carcinogenicagent. A reduction in the severity of a cancer can be indicated by adecrease in size or in growth rate of the tumor, which can be determinedusing various imaging methods. The prevention or reduction in theseverity of a cancer also can be determined indirectly by detecting theactivation of immunoeffector functions in a subject such as theactivation of tumor cytolytic immunoeffector cells.

A composition of the invention can prevent or reduce the severity of acancer by stimulating an immune response against the cancer. As usedherein, the term “stimulating an immune response” means that an immuneresponse is induced or that the activity of immunoeffector cells isenhanced in response to administration to a subject of a composition ofthe invention. Stimulation of an immune response can be indicated bycomparing the immune functions of a subject prior to administration of acomposition with the immune functions following administration. Immunefunctions can be determined using methods described below or otherwiseknown in the art for measuring a humoral or cellular immune response.Prevention or a reduction in the severity of a cancer as indicated bythe methods described herein are an indication that the subject's immuneresponse against the cancer has been stimulated.

In one embodiment, tumor cells are removed from a subject having acancer, which is characterized, in part, by cancer cells that express atleast one immunosuppressive agent such as an immunosuppressive isoformof TGFβ, and the tumor cells are genetically modified to reduce orinhibit the expression of the immunosuppressive agent. Methods forgenetically modifying a cell are known in the art and described indetail below. Although the tumor cells to be genetically modified can beobtained from the subject to be treated, they also can be obtained froma source other than the cancer patient, provided the tumor cells aresubstantially similar to the subject's cancer cells and express animmunosuppressive agent. Tumor cells can be obtained from a source otherthan the subject to be treated if, for example, the subject's own tumorcells are not accessible or the subject is predisposed to, but does notyet have, cancer.

As used herein, the terms “tumor cell” and “cancer cell” are usedinterchangeably to mean a malignant cell. A tumor cell can occur in andcan be obtained from a solid tumor such as a sarcoma, carcinoma,melanoma, lymphoma or glioma or a more diffuse cancer such as aleukemia. Tumor cells can be obtained from a subject having a cancer,from a donor subject having a cancer that is the same or substantiallysimilar to the cancer n the subject to be treated or from a tumor cellrepository. For convenience, the term “donor tumor cell” is used to meantumor cells that are obtained from a source other than the subject to betreated.

It is recognized that a patient's cancer cells may or may not express animmunosuppressive agent. However, only if the patient's cancer cellsexpress one or more immunosuppressive agents are the cancer cellsgenetically modified to reduce or inhibit the expression of theimmunosuppressive agent(s). A subject's cancer cells that do not expressan immunosuppressive factor can be genetically modified to express animmunostimulatory agent as described below.

As used herein, the term “tumor cells that are substantially similar tothe subject's cancer cells” means tumor cells such as allogeneic tumorcells that are of the same or similar histologic type as the subject'scancer cells or that express a tumor specific or tumor associatedantigen that is the same or similar to an antigen expressed by thesubject's cancer cells. Such tumor antigens are known n the art (see,for example, Finn, Curr. Opin. Immunol. 5:701-708 (1993), which isincorporated herein by reference). For convenience of discussion, thesubject's own tumor cells are considered to be within the meaning ofthis term. Allogenic tumor cells that are substantially similar to thesubject's cancer cells can be identified, for example, usinghistological, histochemical, biochemical, molecular biological orimmnological methods well known in the art.

As used herein, the term “immunosuppressive agent” refers to a geneproduct that has an inhibitory effect on the functions of the immuneresponse. An immunosuppressive agent can interfere, for example, withthe function of a cytokine or can inhibit or suppress the immuneresponse by other mechanisms. Immunosuppressive agents are known in theart and include, for example, TGFβ, lymphocyte blastogenesis inhibitoryfactor, the retroviral p15 E protein, suppressive E-receptor (seeSulitzeanu, supra, 1993) and extracellular matrix molecules such asfibronectin and tenascin (Olt et al., Cancer 70:2137-2142 (1992);Hemasath et al., J. Immunol. 152:5199-5207 (1994), each of which isincorporated herein by reference). It is recognized, for examdie, thatvarious isoforms of TGFβ such as TGFβ1, TGFβ2, TGFβ3, TGFβ4 and TGFβ5exist (see, for example, Roszman et al., Immunol. Today, 12:370-274(1991); Constam et al., J. Immunol., 148:1404-1410 (1992); Elliot etal., J. Neuro-Oncoloay, 14:1-7 (1992), each of which is incorporatedherein by reference) and that the immunosuppressive effect of one ormore of these isoforms of TGFβ depends, for example, on the target cell.The term “TGFβ” is used generally herein to mean any isoform of TGFβ,provided the isoform has immunosuppressive activity.

As used herein, the term “express an immunosuppressive agent” means thatthe tumor cells produce an immunosuppressive agent. As used herein, theterm “reduce or inhibit the expression of an immunosuppressive agent” isused in its broadest sense to mean that the level of an RNA moleculeencoding an immunosuppressive agent or the level or activity of theimmunosuppressive agent, itself, is reduced to a level that is less thanthe level expressed prior to the genetic modification. The terms“reduce” and “inhibit” are both used because, n some cases, the level ofexpression of an immunosuppressive agent can be reduced to a level thatis below the level detectable by a particular assay and, therefore, itcannot be determined whether expression of the immunosuppressive agentis reduced or is completely inhibited. Use of the term “reduce orinhibit” prevents any potential ambiguity due, for example, to thelimitations of a particular assay.

Reduction or inhibition of expression of an immunosuppressive agent thatis expressed by a tumor cell can be accomplished using known methods ofgenetic modification. For example, a tumor cell expressing animmunosuppressive agent such as an immunosuppressive isoform of TGFβ canbe genetically modified such that the expression of the TGFβ is reducedor inhibited using a homologous recombination gene “knock-out” method(see, for example, Capecchi, Nature, 344:105 (1990) and references citedtherein; Koller et al., Science, 248:1227-1230 (1990); Zijlstra et al.,Nature, 342:435-438 (1989), each of which is incorporated herein byreference; see, also, Sena and Zarling, Nat. Genet., 3:365-372 (1993),which is incorporated herein by reference). The homologous recombinationgene knock-out method provides several advantages. For example,expression of a gene encoding an immunosuppressive agent such as a TGFβgene in a tumor cell can be inhibited completely if both alleles of thetarget gene are inactivated. In addition to providing completeinhibition of the immunosuppressive agent, the method of homologousrecombination gene knock-out is essentially permanent.

The expression of an immunosuppressive agent by a tumor cell also can bereduced or inhibited by providing in the tumor cell an antisense nucleicacid sequence, which is complementary to a nucleic acid sequence or aportion of a nucleic acid sequence encoding an immunosuppressive agentsuch as an immunosuppressive isoform of TGFβ. Methods for using anantisense nucleic acid sequence to inhibit the expression of a nucleicacid sequence are known in the art and described, for example, by Godsonet al., J. Biol. Chem., 268:11946-11950 (1993), which is incorporatedherein by reference. Expression of an immunosuppressive agent by a tumorcell also can be reduced or inhibited by providing in the tumor cell anucleic acid sequence encoding a ribozyme, which can be designed torecognize and inactivate a specific mRNA such as a mRNA encoding animmunosuppressive isoform of TGFβ (see, for example, McCall et al.,Proc. Natl. Acad. Sci., USA, 89:5710-5714 (1992); Cremisi et al., Proc.Natl. Acad. Sci., USA, 89:1651-1655 (1992); Williams et al., Proc. Natl.Acad. Sci., USA, 89:918-921 (1992); Neckers and Whitesell, Amer. J.Physiol. 265:L1-12 (1993); Tropsha et al., J. Mol. Recog. 5:43-54(1992), each of which is incorporated herein by reference).

The expression of an immunosuppressive agent by a tumor cell also can bereduced or inhibited by genetically modifying the tumor cell to expressa binding protein that can bind the immunosuppressive agent and renderit inactive. For example, the tumor cell can be genetically modified toexpress a natural receptor for the immunosuppressive agent such as aTGFβ2 receptor if the immunosuppressive agent is TGFβ2 or to express anantibody such as a single chain antibody that can specifically bind animmunosuppressive agent in the tumor cell (Duan et al., Proc. Natl.Acad. Sci., USA 91:5075-5079 (1994); Buonocore and Rose, Proc. Natl.Acad. Sci., USA 90:2695-2699 (1993), each of which is incorporatedherein by reference). Expression in the tumor cell of such a bindingprotein can decrease the available immunosuppressive agent and, as aconsequence, reduce or inhibit immunosuppression of a subject's immuneresponse against a cancer.

Various assays to determine whether a subject's cancer cells express animmunosuppressive agent such as an immunosuppressive isoform of TGFβ areavailable and known to those skilled in the art. For example, aradioimmunoassay or enzyme linked immunosorbent assay can be used todetect a specific immunosuppressive agent in a serum or urine sampleobtained from a subject. Other assays such as the mink lung epithelialcell assay can be used, for example, to identify TGFβ2 activity (seeExample I). A biopsy of the tumor can be examined, for example,immunohistochemically for the expression of an immunosuppressive agent.In addition, the tumor cells can be evaluated by northern blot analysis,reverse transcriptase-polymerase chain reaction or other known methods(see, for example, Erlich, PCR Technology: Principles and applicationsfor DNA amplification (Stockton Press 1989); Sambrook et al., MolecularCloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989),each of which is incorporated herein by reference).

As disclosed herein, reducing or inhibiting the expression of animmunosuppressive agent by a tumor cell can attenuate theimmunosuppressive effect of the agent and allow natural immune functionsto work even in the presence of the endogenous circulatingimmunosuppressive agent. Thus, tumor cells that are genetically modifiedto reduce or inhibit the expression of an immunosuppressive agent can beuseful as a vaccine to prevent or reduce the severity of a cancer in asubject by stimulating the subject's immune response against the cancer.For example, tumor cells that are genetically modified by homologousrecombination gene knock-out of a gene encoding an immunosuppressiveagent or by expressing an antisense nucleic acid sequence can beadministered to a cancer patient to stimulate an immune response againstthe cancer and, thereby, reduce the severity of the cancer.

Such genetically modified tumor cells also can be used as a vaccine in asubject predisposed to developing a cancer in order to prevent a cancerfrom occurring in the subject. Such a vaccine can be produced, forexample, by obtaining donor tumor cells that are substantially similarto the suspect cancer. Such donor tumor cells can be obtained, forexample, from a donor subject having a cancer that is the same orsubstantially similar to the suspect cancer and genetically modifyingthe donor tumor cells to reduce or inhibit the expression of animmunosuppressive agent. The genetically modified donor tumor cells canbe administered to the subject alone or in combination with animmunostimulatory agent such as an adjuvant or CE cells in order tostimulate the subject's immune response against the cancer.

In another embodiment, tumor cells are removed from a patient having acancer and genetically modified to reduce or inhibit the expression ofthe immunosuppressive agent and further modified to express and secreteone or more immunostimulatory agents such as a cytokine or a known tumorantigen. The expression of an immunostimulatory agent in combinationwith the removal of the inhibitory effect of an immunosuppressive agentcan augment positive immune functions in a subject. Such immunestimulation can effectively enhance the effect provided by reducing orinhibiting the expression of an immunosuppressive agent in a tumor cell.As a result, the genetically modified tumor cells can be particularlyeffective as a vaccine to prevent or reduce the severity of a cancer ina subject.

As used herein, the term “immunostimulatory agent” is used in itsbroadest sense to mean a molecule that can positively effect theimmunoresponsiveness of a subject. For example, an immunostimulatoryagent can be an adjuvant such as BCG (see Harlow and Lane, Antibodies: Alaboratory manual (Cold Spring Harbor Laboratory Press 1988); Mishelland Shiigi, Selected Methods in Cellular Immunology (W. H. Freeman andCo. (1980)), each of which is incorporated herein by reference) or canbe a gene product that can be administered locally or systemically to asubject or expressed in a cell. A tumor cell or a normal cell such as afibroblast or an antigen presenting cell can be genetically modified toexpress an immunostimulatory agent that is a gene product.Immunostimulatory agents that are gene products are known in the art andinclude, for example, cytokines, the costimulatory B7 molecule (Baskaret al., Proc. Natl. Acad. Sci., USA 90:5687-5690 (1993); Townsend andAllison, Science 259:368-370 (1993); Tan et al., J. Immunol.149:32217-3224 (1992), each which is incorporated herein by reference),autologous MHC class I and class II molecules (Plautz et al., Proc.Natl. Acad. Sci., USA 90:4645-4649 (1993); Hui et al., Fems Microbiol.Immunol. 2:215-221 (1990); Ostrand-Rosenberg et al., J. Immunol.144:4068-4071 (1990), each of which is incorporated herein byreference), alloaeneic histocompatability antigens such as HLA-B7 (Nabelet al., Proc. Natl. Acad. Sci., USA 90:11307-11311 (19931, which isincorporated herein by reference) and known tumor antigens (Finn, supra,1993). For example, a tumor cell may not express an MHC class I or IImolecule and, as a result, does not induce an optimal immune response.In this case, the MHC molecule can be an immunostimulatory agent, since,by expressing the MHC molecule in the tumor cell, the modified tumorcell can induce an immune response. Methods for determining whether atumor cell expresses a particular immunostimulatory agent are known inthe art and can be used to determine whether the tumor cell should begenetically modified to express a particular immunostimulatory agent.

A known tumor antigen can be particularly useful as an immunostimulatoryagent. Various tumor antigens, including, for example, epithelial cellmucin, which is encoded by the MUC-1 gene, and the melanoma antigen,MZ2-E, which is encoded by the MAGE-1 gene, are associated withparticular tumor cells (Finn, supra, 1993). Genetically modifying atumor cell to express a known tumor antigen can be particularly usefulwhen the tumor cells to be administered to a subject to be treated arenot obtained from that subject. For example, it may not be possible toobtain a sufficient number of tumor cells from a cancer patient. In thiscase, donor tumor cells, which may not express one or more particulartumor antigens that are known to be expressed by the subject's cancercells, can be obtained and can be genetically modified to express theparticular tumor antigen. The donor tumor cells also will be geneticallymodified to reduce or inhibit the expression of an immunosuppressiveagent. Upon administration of the genetically modified donor tumor cellsto the subject, the subject's immune response against the cancer can bestimulated against the subject's cancer. Such genetically modified donortumor cells also can be useful as a vaccine to prevent the developmentof a cancer in a subject predisposed to developing a particular cancer.

A cytokine can be useful as an immunostimulatory agent. As used herein,the term “cytokine” refers to a member of the class of proteins that areproduced by cells of the immune system and positively regulate ormodulate effector functions of the immune response. Such regulation canoccur within the humoral or the cell mediated immune response and canmodulate the effector functions of T cells, B cells, macrophages,antigen presenting cells or other immune system cells. Specific examplesof cytokines include, for example, interleukin-1, interleukin-2,interleukin-3, interleukin-4, interleukin-5, interleukin-6,interleukin-7, interleukin-10, interleukin-12, interleukin-15,gamma-interferon, tumor necrosis factor, granulocyte colony stimulatingfactor and granulocyte-macrophage colony stimulating factor.

It is recognized that the expression of specific combinations ofcytokines can be particularly useful for stimulating an immune response.For example, expression of gamma-interferon, IL-2 and interleukin-12 canstimulate T cells of the T helper-1 class, which are involved in thecellular immune response. Thus, it can be particularly useful togenetically modify tumor cells to reduce or inhibit the expression ofone or more immunosuppressive agents, then to individually furthermodify aliquots of the cells to express gamma-interferon or IL-2 orinterleukin-12. A composition comprising a combination of suchgenetically modified tumor cells can be administered to a subject tostimulate, in particular, a cellular immune response against the tumorcells.

In some cases, it can be difficult to obtain a sufficient number ofcancer cells from a patient. Donor tumor cells that are geneticallymodified to reduce or inhibit the expression of an immunosuppressiveagent and, if desired, further modified to express a known tumor antigenor a cytokine can be used to stimulate an immune response in such apatient. The genetically modified donor tumor cells also can be used toprevent a cancer .In a normal subject or in a subject suspected ofdeveloping a cancer. Subjects that are predisposed to developing acancer are known and can be identified using methods of geneticscreening (Mao et al., Canc. Res. 54(Suppl.):1939s-1940s (1994); Garberand Diller, Curr. Opin. Pediatr. 5:712-715 (1993). Such subjects can bepredisposed to developing, for example, retinoblastoma, breast cancer orcolon cancer.

A panel of genetically modified donor tumor cells, which can representvarious histologic tumor types and express various known tumor antigenssuch as MZ2-E or mucin (see Finn, supra, 1993), can be prepared. Such apanel of genetically modified donor tumor cells can be maintained in acell repository and are readily available for administration to asubject predisposed to developing a particular cancer. One or more tumorcell lines in such a panel can be used to stimulate a cancer patient'simmune response against the patient's cancer. The skilled artisan canselect an appropriate genetically modified donor tumor cell from thepanel based, for example, on the histologic type of tumor the subjecthas or is predisposed to developing. If desirable, the artisan canfurther genetically modify a tumor cell obtained from such a paneldepending on the particular characteristics of the cancer in the subjectto be treated.

In still another embodiment, cancer cells are removed from a patienthaving a cancer, which is characterized, in part, by expressing animmunosuppressive agent, or donor tumor cells are obtained and the cellsare genetically modified to reduce or inhibit the expression of theimmunosuppressive agent. The genetically modified tumor cells then arecombined with an immunostimulatory agent such as CE cells or an adjuvantto provide a composition that can be used to stimulate the cancerpatient's immune response.

As used herein, the term “CE cell” or “cytokine-expressing cell” means acell such as a fibroblast or an antigen presenting cell that isgenetically modified to express and secrete one or more cytokines. Asdescribed above, the use of the cytokine gene therapy can augmentpositive immune functions in a subject in combination with therepression of the inhibitory effects of an immunosuppressive agent. A CEcell can be an autologous cell, which is obtained from the subject to betreated, or can be an allogeneic cell, which can be obtained, forexample, from a donor subject or from a cell repository. It isrecognized that cells to be used as CE cells must be examined todetermine whether they express an immunosuppressive agent. If the cellsexpress an immunosuppressive agent, they can be genetically modified toreduce or inhibit the expression of the agent using the methodsdescribed herein.

The invention also provides methods for preventing or reducing theseverity of a cancer in a subject. Such a method can consist, forexample, of stimulating the subject's immune response against the cancerby administering to the subject an effective amount of tumor cells thatare substantially similar to the subject's cancer cells and that aregenetically modified to prevent the expression of at least oneimmunosuppressive agent. In addition, the genetically modified tumorcells can be further modified to express and, if desirable, secrete animmunostimulatory agent or can be administered in combination with animmunostimulatory agent such as an adjuvant or an effective amount of acytokine or of CE cells (see, for example, Khan et al., Pharm. Res.11:2-11 (1994); Audibert and Lise, Immunol. Today 4:281-284 (1993), eachof which is incorporated herein by referenced.

As used herein, the term “effective amount” means an amount of asubstance such as the genetically modified tumor cells, alone, or incombination with an immunostimulatory agent such as a cytokine or CEcells, that can stimulate an immune response to prevent or reduce theseverity of a cancer in a subject. Such an effective amount can bedetermined using assays for determining the activity of immunoeffectorcells following administration of the substance to the subject or bymonitoring the effectiveness of the therapy using well known in vivodiagnostic assays as described below.

Based merely on in vitro experiments, it previously was suggested thatthe expression of TGFβ by a tumor cell inhibited immunoeffectorfunctions against the tumor cell and that the inhibition of TGFβexpression in such tumor cells would render the cells more susceptibleto immunoeffector cells (Hachimczak et al., J. Neurosura. 78:944-951(1993)). However, in vitro experiments to examine theimmunoresponsiveness of immunoeffector cells against tumor cellsfollowing a particular treatment in vitro is not necessarily predictiveof in vivo efficacy of the therapy. For example, rats were immunizedwith 9L gliosarcoma cells or 9L cells that had been genetically modifiedto express IL-2. When anti-tumor lytic activity of lymph node effectorcells was examined in vitro, it was observed that in vitro-stimulatedimmunoeffector cells obtained from the rats immunized with thegenetically modified gliosarcoma cells had greater cytolytic activityagainst target 9L gliosarcoma cells and greater natural killer cellactivity than in vitro-stimulated immunoeffector cells obtained fromunimmunized rats or rats immunized with unmodified 9L gliosarcoma cells(FIG. 1; see, also, Example I). If such in vitro results were predictiveof in vivo efficacy, it would have been expected that immunization ofrats with the IL-2 modified gliosarcoma cells would produce a greaterimmune response in the rats against 9L tumor cells. However, nosignificant difference in animal survival was observed in rats that wereinjected with 5×10³ 9L gliosarcoma cells on days 1 and 2, then immunizedtwice per week for two weeks, beginning on day 5, with unmodified orwith IL-2 modified 9L cells (see Table, below). Furthermore, previous invitro studies showed that IL-2 and antibodies to TGFβ couldsignificantly counteract TGFβ-induced depression n lymphocytes. However,when administered in vivo, these treatments resulted in either noanti-tumor effects or enhanced tumor growth (Gridley et al., Canc.Biother. 8:159-170 (1993)). Thus, in vitro results are not necessarilypredictive of in vivo efficacy of a treatment.

Moreover, although expression of an antisense TGFβ nucleic acid sequence(“antisense TGFβ”) in 9L gliosarcoma cells rendered the gliosarcomacells immunogenic in vivo (see Table I, below), this was not the case inanother animal model. Specifically, when antisense TGFβ was expressed inovarian carcinoma cells, the genetically modified cells were no moreimmunogenic than the ovarian carcinoma cells administered alone or incombination with IL-2-producing CE cells (see FIG. 2). However, when acomposition of ovarian tumor cells that had been genetically modified toexpress antisense TGFβ was administered to an experimental animal incombination with IL-2-producing CE cells, an immune response wasstimulated against the tumor cells. Thus, as disclosed herein, thepresent invention provides methods for stimulating an immune response ina subject against the subject's cancer cells by immunizing the subjectwith tumor cells, which are genetically modified to prevent expressionof an immunosuppressive agent, alone or in combination with animmunostimulatory agent.

The methods of tumor immunotherapy described herein utilize the geneticmodification of tumor cells, which results in the reduction orinhibition of expression of one or more immunosuppressive agents by thetumor cells. Homologous recombination gene knock-out is particularlyuseful for reducing or inhibiting the expression of an immunosuppressiveagent because it results in essentially total and permanent inhibitionof expression of the immunosuppressive agent. Expression of an antisensenucleic acid sequence also can reduce or inhibit the expression of theimmunosuppressive agent by the tumor cell. Regardless of the method forreducing or inhibiting expression of an immunosuppressive agent by atumor cell, administration of the genetically modified tumor cells to asubject can stimulate a subject's immune response against a cancer.Thus, the disclosed methods are useful for reducing the severity of anexisting tumor in a subject or for preventing the occurrence of a cancerin a subject predisposed to developing a cancer.

Tumor cells that show enhanced efficacy as an anti-tumor vaccine due toreduction or inhibition of expression of one or more immunosuppressiveagents can be used therapeutically to treat a subject for a particularcancer. Tumor cells to be genetically modified can be obtained, forexample, by biopsy from the subject having the cancer, which ischaracterized, in part, by expressing one or more immunosuppressiveagents, and the tumor cells can be genetically modified to inhibit theexpression of the immunosuppressive agent(s). Alternatively, asdescribed above, donor tumor cells can be obtained and geneticallymodified. The genetically modified tumor cells then can be administeredto the subject.

It is recognized that the tumor cells to be administered should beviable. However, administration of viable tumor cells to a subjectrequires that the tumor cells be inactivated so they do not grow in thesubject. Inactivation can be accomplished by any of various methods,including, for example, by irradiation, which is administered to thecells at a dose that inhibits the ability of the cells to replicate butdoes not initially kill the tumor cells. Such viable tumor cells canpresent tumor antigens to the patient's immune system but cannotmultiply and form new tumors.

Once the tumor cells are obtained from either the subject to be treated,a donor subject or an established cell line, the tumor cells aregenetically modified such that expression of one or moreimmunosuppressive agents expressed by the tumor cell is reduced orinhibited (see Example II). Genetic modification is advantageous overother methods for inhibiting expression of the immunosuppressive agentin that it is efficient, specific for the intended gene product and canprovide sustained inhibition of the expression of one or moreimmunosuppressive agents. As described above, methods of geneticmodification include, for example, homologous recombination, which canpermanently and completely inactivate the gene encoding theimmunosuppressive factor, or expression of a ribozyme or an antisensenucleic acid sequence in a cell, which can inhibit or inactivate one ormore steps involved in transcription, processing or translation of anucleic acid molecule encoding the immunosuppressive agent. In addition,genetic modification can result in the expression in the tumor cell of abinding protein such as an antibody, which can specifically bind to animmunosuppressive agent and prevent the immunosuppressive activity.

Homologous recombination gene knock-out is an effective method ofreducing or inhibiting expression of an immunosuppressive agent becauseof the complete and sustained inhibition of expression of the agent. Inaddition, antisense methods for reducing or inhibiting expression of animmunosuppressive agent are useful as disclosed herein. Antisensemethods involve introducing into the tumor cell a nucleic acid sequencethat is complementary to and can hybridize to the target nucleic acidmolecule, which encodes the immunosuppressive agent, in a cell. Anantisense nucleic acid sequence can be a chemically synthesizedoligonucleotide, which can be introduced into the tumor cells by methodsof transfection, or can be expressed from a vector, which can be stablyintroduced into the tumor cell using well known methods (see, forexample, Sambrook et al., supra, 1989). One in the art would know thatthe ability of such a complementary nucleic acid sequence to hybridizeto the target nucleic acid sequence depends, for example, on the degreeof complementarity shared between the sequences, the length of theantisense nucleic acid sequence, which generally is an oligonucleotidethat is at least ten nucleotides in length, and the GC content of theoligonucleotide (see Sambrook et al., supra, 1989).

A recombinant vector can be used to express an antisense nucleic acidsequence in the tumor cells. Such vectors are known or can beconstructed by those skilled in the art and contain the expressionelements necessary to achieve, for example, sustained transcription ofan antisense nucleic acid sequence. Examples of vectors include virusessuch as bacteriophages, baculoviruses and retroviruses and DNA viruses,cosmids, plasmids or other recombination vectors (Jolly, Canc. GeneTher. 1:51-64 (1994), which is incorporated herein by reference). Thevectors can also contain elements for use in procaryotic or eucaryotichost systems or, if desired, both systems. One of ordinary skill in theart would know which host systems are compatible with a particularvector. Vectors that result in high levels of sustained expression ofthe antisense nucleic acid sequences can be particularly useful.

Examples of useful viral vectors, for example, include adenovirus andadenovirus-associated vectors (see, for example, Flotte, J. Bioenera.Biomemb., 25:37-42 (1993) and Kirshenbaum et al., J. Clin. Invest,92:381-387 (1993), each of which is incorporated herein by references.Vectors are particularly useful when the vector contains a promotersequence, which can provide constitutivector, if desired, inducibleexpression of a cloned nucleic acid sequence. Such vectors are wellknown in the art (see, for example, Meth. Enzymol., Vol. 185, D. V.Goeddel, ed. (Academic Press, Inc., 1990), which is incorporated hereinby reference) and available from commercial sources (Promega, Madison,Wis.).

Vectors can be introduced into the tumor cells by any of a variety ofmethods known in the art and described, for example, in Sambrook et al.,supra, 1989, and in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1994), which isincorporated herein by reference. Such methods include, for example,transfection, lipofection, electroporation and infection withrecombinant vectors or the use of liposomes. Introduction of nucleicacids by infection is particularly advantageous in that it can beeffective in vitro or in vivo. Higher efficiency can also be obtaineddue to their infectious nature. Moreover, viruses are very specializedand typically infect and propagate in specific cell types. Thus, theirnatural specificity can be used to target the antisense vectors tospecific tumor cell types in a biopsy culture, which may be contaminatedwith other cell types. Viral or non-viral vectors can also be modifiedwith specific receptors or ligands to alter target specificity throughreceptor mediated events. The use of retroviral vectors for theexpression of antisense sequences is described below.

A nucleic acid molecule also can be introduced into a tumor cell usingmethods that do not require the initial introduction of the nucleic acidsequence into a vector. For example, a nucleic acid sequence useful forinactivating a gene in a tumor cell such as a gene encoding animmunosuppressive isoform of TGFβ can be introduced into a tumor cellusing a cationic liposome preparation (Morishi-a et al., J. Clin.Invest., 91:2580-2585 (1993), which is incorporated herein by reference;see, also, Nabel et al., supra, 1993)). In addition, a nucleic acidsequence can be introduced into a tumor cell using, for example,adenovirus-polylysine DNA complexes (see, for example, Michael et al.,J. Biol. Chem., 268:6866-6869 (1993), which is incorporated herein byreference). Other methods of introducing a nucleic acid sequence into atumor cell such that the nucleic acid can be expressed or can result inthe reduction or inhibition of expression of a gene product are wellknown and described, for example, by Goeddel, supra, 1990).

Nucleic acid sequences encoding various immunosuppressive andimmunostimulatory agents have been cloned and are available for use(GenBank). Nucleic acid sequences encoding, for example, cytokines suchas various interleukins, gamma-interferon and colony stimulating factorsare available from the American Type Culture Collection (see ATCC/NIHRepository Catalogue of Human and Mouse DNA Probes and Libraries, 6thed., 1992) or are available commercially (Amgen, Thousand Oaks, Calif.;see, also, Patchen et al., Exptl. Hematol., 21:338-344 (1993); Broudy etal., Blood, 82:436-444 (1993), each of which is incorporated herein byreference). Similarly, nucleic acid sequences encoding immunosuppressiveagents such as various immunosuppressive isoforms of TGFβ are availableto those in the art.

In addition, a nucleic acid sequence encoding an immunosuppressiveagent, for example, can be obtained and modified such that, followinghomologous recombination with the target gene in the tumor cell, theimmunosuppressive agent is not expressed. Nucleic acid sequencesencoding various immunosuppressive agents or immunostimulatory agentsalso can be obtained using, for example, the polymerase chain reaction,provided some information of the nucleic acid sequence encoding theagent is known. Other methods such as cloning also can be used toisolate a desired nucleic acid sequence encoding an immunosuppressive orimmunostimulatory agent.

Selectable marker genes encoding, for example, a polypeptide conferringneomycin resistance (Neo^(R)) also are readily available and, whenlinked to a nucleic acid sequence or incorporated into a vector, allowsfor the selection of cells that have successfully incorporated thedesired nucleic acid sequence, which can inactivate a target gene byhomologous recombination, encode an antisense nucleic acid sequence orencode an immunostimulatory agent. Other selectable markers known tothose in the art of gene transfer also can be used to identify tumorcells that have been genetically modified, for example, by homologousrecombination to reduce or inhibit the expression of one or moreimmunosuppressive factors.

A “suicide” gene also can be incorporated into a vector so as to allowfor selective inducible killing of a genetically modified tumor cellfollowing stimulation of a subject's immune response. A gene such as theherpes simplex virus thymidine kinase gene (TK) can be used as a suicidegene to provide for an inducible destruction of the tumor cells. Forexample, when the tumor cells are no longer useful, a drug such asacyclovir or gancyclovir can be administered to the subject. Either ofthese drugs selectively kills cells expressing a viral TK, thuseliminating the implanted genetically modified tumor cells.Additionally, a suicide gene can encode a non-secreted cytotoxicpolypeptide and can be linked to an inducible promotor. If destructionof the tumor cells is desired, the appropriate inducer of the promotoris administered so that the cytotoxic polypeptide is expressed.

Numerous methods are available for transferring nucleic acid sequencesinto cultured cells, including the methods described above. in addition,a useful method can be similar to that employed in previous human genetransfer studies, where tumor infiltrating lymphocytes (TILs) weremodified by retroviral gene transduction and administered to cancerpatients (Rosenberg et al., New Engl. J. Med. 323:570-578 (1990)). Inthat Phase I safety study of retroviral mediated gene transfer, TILswere genetically modified to express the Neomycin resistance (Neo³)gene.Following intravenous infusion, polymerase chain reaction analysesconsistently found genetically modified cells in the circulation for aslong as two months after administration. No infectious retroviruses wereidentified in these patients and no side effects due to gene transferwere noted in any patients. These retroviral vectors have been alteredto prevent viral replication by the deletion of viral gag, pol and envgenes. Such a method can also be used ex vivo to transduce tumor cellstaken from a subject with cancer.

When retroviruses are used for gene transfer, replication competentretroviruses theoretically can develop due to recombination ofretroviral vector and viral gene sequences in the packaging cell lineutilized to produce the retroviral vector. Packaging cell lines in whichthe production of replication competent virus by recombination has beenreduced or eliminated can be used to minimize the likelihood that areplication competent retrovirus will be produced. Hence, all retroviralvector supernatants used to infect patient cells will be screened forreplication competent virus by standard assays such as PCR and reversetranscriptase assays.

Approximately 1×10⁷ tumor cells are required for immunization,depending, for example, on the number of times the composition is to beadministered and the level of response desired. The tumor cells can bemixed with an appropriate adjuvant or with a pharmacologicallyacceptable solution such as physiological saline or the like foradministration, which can be accomplished by any of various methods suchas subcutaneous or intramuscular injection or any manner acceptable forimmunization. Pharmacologically acceptable solutions useful forimmunization are known in the art (see, for example, Khan et al., supra,1994; Audibert and Lise, supra, 1993; Mishell and Shiigi, supra, 1980).In addition, various methods of administration can be used and are knownto those skilled in the art. Administration can be at a body locationother than an active tumor site or, if desired, at the site of a tumorin a cancer patient or can be in a subject predisposed to developing acancer.

One skilled in the art would know that the effectiveness of therapy canbe determined by monitoring immune functions in the patient. Forexample, the cytolytic activity of immune effector cells against thepatient's cancer cells can be assayed using the methods described inExample I. In addition, the size or growth rate of a tumor can bemonitored in vivo using methods of diagnostic imaging. By monitoring thepatient during therapy, the therapist would know whether to maintain,for example, therapy using tumor cells that are genetically modified toprevent expression of an immunosuppressive agent or whether it is moredesirable to use a combination therapy, which can reduce or prevent theexpression of an immunosuppressive agent and, at the same time, canprovide an immunostimulatory agent. Such combined gene therapy canexhibit superior efficacy because not only are the immunosuppressivefunctions depressed but the stimulatory functions of the immune systemare enhanced. This combined approach is particularly useful whenseparate inhibition of an immunosuppressive agent or stimulation of theimmune response using an immunostimulatory agent provides only moderateeffects when used alone.

Where combined therapy is used, tumor cells expressing, for example, anantisense nucleic acid sequence to an immunosuppressive agent can befurther modified to express one or more immunostimulatory agents such ascytokines. The tumor cells should be modified so that a known number ofcells in the composition secrete, for example, appropriate cytokinelevels, which can induce anti-tumor immunity without producingsubstantial systemic toxicity in a subject. Alternatively, thegenetically modified tumor cells can be administered in combination withCE cells, which express and secrete a defined level of cytokines. Theexpression of an appropriate level of a cytokine allows administrationof an effective amount of the composition, while minimizing thelikelihood of adverse side effects as were observed using previouslydescribed methods of systemic administration of greater thanphysiological levels of the cytokines.

As with the tumor cells modified with antisense described previously,tumor cells that express an antisense nucleic acid and animmunostimulatory agent or the combination of tumor cells that expressan antisense nucleic acid sequence for an immunosuppressive agent and animmunostimulatory agent such as a cytokine or CE cells can be formulatedfor injection in any manner known in the art as being acceptable forimmunization. Because it is important that the tumor cells and, if used,CE cells remain viable, the formulations are compatible with cellsurvival. It is recognized that contamination of a composition of theinvention can focus the immune response on undesired antigens and,therefore, should be avoided by practicing aseptic techniques.

It is understood that modifications that do not substantially affect theactivity of the embodiments of this invention also are included withinthe invention provided herein. Accordingly, the following examples areintended to illustrate but not limit the present invention.

EXAMPLE I IMMUNOSUPPRESSIVE EFFECT OF TGFβ IN TUMOR CELLS

This example demonstrates that various tumor cell lines produce animmunosuppressive isoform of TGFβ.

A murine ovarian teratoma cell line, MOT D3-17-5 (MOT), a humanglioblastoma cell line, GT-9, and a rat gliosarcoma cell line, 9L, wereused to assess the immunosuppressive effects of TGFβ. Expression of TGFβwas determined by measuring the ability of TGFβ to suppress the growthof mink Mv-1-Lu lung epithelial cells (ATCC CCL64; Rockville Md.; Ogawaand Seyedin, Meth. Enzymol. 198:317-327 (1991), which is incorporatedherein by reference). Briefly, mink lung epithelial cells are grown onDMEM containing 10% FCS, 50 U/ml penicillin, 50 μg/ml streptomycin,nonessential amino acids and L-glutamine until near confluency. Cellswere trypsinized and collected by centrifugation at 800×g for 2 min,then resuspended in culture medium at 20,000 cells/ml. The cells wereplated on 96-well microtiter plates at 1000 cells/well (50 ul/well) andallowed to attach for 30 min.

Fifty μl conditioned supernatant from the various cultured tumor celllines were added in triplicate to well containing mink lung epithelialcells. Plates were incubated for 4 days at 37° C. in an atmospherecontaining 10% CO₂. Wells were rinsed with phosphate buffered saline(PBS), then filled with 100 ul 0.1 M sodium acetate (pH 5.5)/0.1% TritonX-100/100 mM p-nitrophenylphosphate and the plates were incubated for 2hr at 37° C. Color development was generated by the addition of 10 ul1.0 N NaOH and the absorbance at 405 nm, which is proportional to thenumber of cells, was determined. Supernatants obtained from the MOT,GT-9 and 9L tumor cell cultures inhibited mink lung epithelial cellproliferation in a dose-dependent manner, indicating that each of thesetumor cell lines secrete biologically active TGFβ (not shown).

The mink lung epithelial cell assay also can be performed using minklung epithelial cells that are pulsed for 4 hr with 0.5 μCi³H-thymidine. In this case, the number of counts is proportional to thenumber of proliferating cells, which decreases in the presence ofincreasing amounts of TGFβ.

The specific isoform of TGFβ in a sample can be determined usinganti-TGFβ antibodies. However, since TGFβ exists in a cell in an activeform and in an inactive form and since anti-TGFβ antibody only reactswith active TGFβ, in some experiments, TGFβ can be activated byacidification so that total TGFβ in the sample can be determined. Priorto addition of a cell supernatant to the mink lung epithelial cells, thesupernatants either are left untreated, in order to measure the amountof active TGFβ secreted, or are acidified to activate TGFβ. The culturesupernatant is acidified by adjusting to pH 2-3 with HCl, incubated for30 min, then neutralized with NaOH. Antibodies specific for TGFβ1 orTGFβ2 (R & D Systems; Minneapolis Minn.) are added to some wells todetermine specificity. Known concentrations of TGFβ are used to generatea standard curve.

To determine the inhibitory effect of TGFβ on immunoresponsiveness usingthe 9L tumor model, F344 rats were immunized subcutaneously with 2×10⁶irradiated (5000 cGy) unmodified 9L gliosarcoma cells or 9L cells thathad been transduced with LNCX-IL2, which is a retroviral vector thatexpresses IL-2. The LNCX-IL2 vector was constructed by inserting a humanIL-2 cDNA containing the rat preproinsulin secretory signal (Cullen, DNA7:645-650 (1988), which is incorporated herein by reference into LNCX,which contains a cytomegalovirus (CMV) promotor and was packaged inPA317 cells (Miller and Buttimore, Mol. Cell Biol. 6:2895-2902 (1986),which is incorporated herein by reference).

For transduction, 9L cells were seeded at a density of 2×10⁶ cells/T-75flask (20% confluency). After the cells attached to the flask, thesupernatant was removed and the cultures were washed once with PBS, thenincubated at 37° C. in serum-free medium containing 20 μg/mlDEAE-dextran. After 30 min, the DEAE-dextran was removed and replacedwith 10 ml medium containing LNCX-IL2 and 8 μg/ml polybrene. To provideadequate nutrient levels during the transduction period, the retroviralsupernatant was diluted 1:1 with fresh medium before being adjusted to 8μg/ml polybrene. The virus supernatant was removed from cultures after 6to 24 hr and replaced with fresh medium. Selection of transductants wasinitiated with 50-400 μg/ml active G418 beginning the next day.

Fourteen days following immunization of the rats with unmodified ortransduced 9L gliosarcoma cells, the lymph nodes were removed and lymphnode effector cells were isolated, then incubated for 5 days in DMEMsupplemented with 10% fetal calf serum (FCS) and 50 U/ml recombinanthuman IL-2. Irradiated 9L cells (5000 cGy) were added at an effectorcell:stimulator cell ratio of 30:1 to duplicate flasks. Turkey anti-TGFβantiserum was added to one flask.

After 5 days, the splenocytes or lymph node cells were harvested andassayed for lytic activity in vitro by a four hour chromium releaseassay using ⁵¹Cr-labeled 9L cells as target cells (Shawler et al., J.Clin. Lab. Anal. 1:184-190 (1987); Dillman et al., J. Immunol.136:728-731 (1986), each of which is incorporated herein by reference.In addition, ⁵¹Cr-labeled A2T2C4 cells, which are a rat naturalkiller-(NK-) sensitive cell line, (Gallinmore et al., J. Mol. Biol.89:49-72 (1974), which is incorporated herein by reference) were used astargets to test for NK activity.

The results of these assays demonstrated that cytotoxic T cell activityand NK cell activity was significantly greater in rats immunized with 9Lcells that had been genetically modified to express IL-2 as compared tounmodified 9L cells. The addition of neutralizing concentrations ofanti-TGFβ2 antibody to the in vitro stimulation portion of the assaycaused about a two-fold increase in the killing of 9L gliosarcoma targetcells at an effector cell:target cell ratio of 100:1 (not shown).However, no significant increase in animal survival time was observed inrats immunized with the genetically modified 9L cells as compared tounimmunized rats or rats immunized with 9L cells transduced with acontrol vector.

The results of these experiments indicate that TGFβ expression canreduce or prevent an immune response to a cancer in vivo. In addition,the results indicate that the effectiveness of a immunostimulatory agentsuch as IL-2 at inducing immunoresponsiveness as determined using an invitro assay is not necessarily predictive of the ability of the agent tostimulate an immune response in vivo.

EXAMPLE II EFFECT OF ANTISENSE TGFβ EXPRESSION ON IMMUNOGENICITY OFTUMOR CELLS

This example demonstrates that expression of an antisense TGFβ nucleicacid sequence in a tumor cell, which is characterized, in part, byproducing TGFβ, allows for the induction of an immune response againstthe tumor cells in vivo.

A. 9L Gliosarcoma Cells

To analyze the systemic anti-tumor effects of inhibiting animmunosuppressive agent such as TGFβ, 9L gliosarcoma cells weretransfected with a vector expressing an antisense TGFβ nucleic acidsequence. A nucleic acid sequence encoding antisense TGFβ2 was insertedinto the pCEP4 vector (Invitrogen; San Diego Calif.). The TGFβ2 nucleicacid sequence was obtained from pSTGFbeta2, which contains a cDNAencoding TGFβ2 that was isolated from African green monkey kidney cells(Hanks et al., Proc. Natl. Acad. Sci., USA 85:79-82 (1988), which isincorporated herein by reference; ATCC #77322; Rockville Md.)

pSTGFbeta2 was digested with XhoI and HindIII to release a 957 base pair(bp) fragment, which contains 870 bp encoding the 5′ end of TGFβ2. Theexpression vector pCEP4 also was digested with HindIII and XhoI. Therestriction enzyme digested products were fractionated byelectrophoresis in an agarose gel, the bands corresponding to thelinearized pCEP4 vector and the 957 bp TGFβ2 sequence were removed andthe nucleic acids were released from the gel slices using the routineglass powder method (see Sambrook et al., supra, 1989). The isolatedvector and the TGFβ2 fragment were ligated overnight at 16° C. and theligation product was used to transform XL-1 bacteria. A clone containinga plasmid having the appropriate sized band was isolated and used forlarge scale DNA preparation (pCEP4/TGFB2). Ligation of the XhoI/HindIIIdigested 957 bp TGFβ2 fragment into pCEP4 placed the TGFβ2 fragment in areverse orientation with respect to the CMV promotor in pCEP4.

pCEP4/TGFB2, which encodes antisense TGFβ2, was transfected using thecalcium phosphate method into 9L gliosarcoma cells and into 9L cellsthat were transduced to contain an IL-2 expressing retroviral vector(91/LNCX/IL2 cells) or a control retroviral vector (9L/LXSN/O cells).The transfected cells were cultured in presence of hygromycin, whichselects for cells containing pCEP4/TGFB2, until colonies appeared.

TGFβ secretion by the transfected cell lines was determined using themink lung epithelial cell assay. Cells that contained pCEP4/TGFB2 werenegative for TGFβ activity (not shown). These results indicate that anantisense TGFB2 produced in the transfected cells can hybridize with anucleic acid molecule encoding TGFβ in the cell and reduce or inhibitthe expression of TGFβ.

The cytolytic activity of immune effector cells in vitro was examined ina 4 hour ⁵¹Cr release assay as described above. Briefly, lymph nodesfrom rats immunized with unmodified 9L cells or with 9L cells modifiedto express and secrete IL-2 were collected and lymph node cells culturedfor 5 days in presence of 50 BRMP units IL-2 per ml and eitherunmodified 9L cells or antisense TGFB2 modified 9L cells. Cytotoxicityof the effector cells was determined in a 4 hour ⁵¹Cr release assayusing unmodified 9L cells as targets. As shown in FIG. 1, lymph nodecells stimulated in vitro by 9L cells expressing antisense TGFB2exhibited significantly increased cytolytic activity against 9L cellsthan effector cells cultured in the presence of unmodified 9L cells.

The effect of immunization on the survival of rats having intracraniallyimplanted 9L gliosarcoma cells also was determined. 5×10³ 9L cells wereimplanted into the forebrain of rats on days 1 and 2. Beginning on day5, the rats were immunized four times on a twice per week schedule witheither saline, 9L cells transduced with a control vector, 9L cellsgenetically modified to express antisense TGFβ2, 9L cells transduced toexpress IL-2 or 9L cells genetically modified to express antisense TGFβ2and IL-2.

As shown in the Table, immunization with unmodified 9L cells (control)or 9L cells expressing IL-2 resulted in survival of 30% of the immunizedanimals. Significantly, the survival of rats immunized with 9L cellstransduced to express IL-2 was the same as rats immunized withunmodified 9L cells. In contrast, 100% survival was observed in ratsimmunized with 9L cells that express antisense TGFβ2, regardless ofwhether the 9L cells also expressed IL-2. These results indicate thatthe expression of antisense TGFβ2 in a tumor cell that expresses TGFβcan render the tumor cells immunogenic in tumor bearing host.

A retroviral vector encoding an antisense TGFβ2 also was constructedusing the retroviral parental plasmid pLHCX, which is a modification ofpLNCX in which the gene conferring neomycin drug resistance is replacedwith a gene conferring hygromycin resistance. pLHCX was digested withHindIII and HpaI and the linearized plasmid was purified from an agarosegel as described above. pSTGFbeta2 was digested with XhoI and the XhoIend was blunted by treatment with Klenow fragment of E. coli DNApolymerase I in presence of all four deoxynucleotides. After phenol

TABLE Effect of Immunization with IL-2 and TGF-βas Modified Tumor Cellson Survival of Rats with Intracranially Implanted Tumors Animals AntigenUnits IL-2 surviving Inoculum dose per 24 hr (11 weeks) Unimmunized — 00/10 9L/Vector (control) 2.5 × 10⁵ 0 3/10 9L/IL-2 2.5 × 10³ 50  3/109L/TGF-βas 2.5 × 10⁵ 0 6/6  9L/TGF-βas/IL-2 2.5 × 10³ 50  7/7  — 5 × 10³tumor cells were implanted into the forebrain of rats on days 1 & 2.Beginning on day 5 animals were immunized 4 times on a twice a weekschedule

extraction and ethanol precipitation the linearized pSTGFbeta2 wasdigested with HindIII to release a 957 bp band, which contains 870 bp ofthe 5′ end of the TGFβ2 cDNA. Transduction of the tumor cells wasperformed as described above and transduced tumor cells selected bygrowth in the presence of hygromycin.

Successful inhibition of TGFβ expression in the modified tumor cells wasconfirmed by determining TGFβ levels in the supernatants of the modifiedcell cultures using the mink cell assay described above. Transgeneexpression also can be assessed at the RNA level using RNA isolated bythe method of Chomczynski and Sacchi (Anal. Biochem., 162:156-159(1987), which is incorporated herein by reference). Briefly, transducedcells are lysed using lysis buffer containing 4 M guanidiniumthiocyanate, then the lysate is extracted using water-saturated phenoland chloroform to remove the cellular proteins. RNA is isolated byisopropanol precipitation and used to synthesize first strand cDNA formRNA PCR analysis or used for northern blot analysis. First strand cDNAis synthesized using the first strand Cycle™ kit according to themanufacturer's recommendation (Invitrogen; San Diego Calif.).

In order to determine the level of in vitro transgene expression, RNA isisolated from cells that are recovered following subcutaneous injectioninto rats and is subjected to PCR analysis (Mullis and Faloona, Meth.Enzymol., 155:335-350 (1987), which is incorporated herein byreference). First strand cDNA is synthesized by the standard primerextension of oligo dT-primed mRNA using reverse transcriptase. PCRamplification is performed in 100 μg/ml nuclease-free BSA, 35 200 μMeach DATP, dGTP, dCTP and dTTP, template-DNA, 100 pmol each primer and2.5 units Taq polymerase. Temperatures and cycle numbers that minimizefortuitous bands are selected for these PCR amplifications (Hickey etal., J. Exp. Med. 176:811-187 (1992), which is incorporated herein byreference).

Transcription efficiency is determined by northern blot analysis. TotalRNA or mRNA aliquots are fractionated by electrophoresis in an agarosegel containing formaldehyde, then transferred to a nylon membrane(Fakhrai and Mins, J. Biol. Chem. 267:4023-4029 (1992); Morrissey etal., Cell 50:129-135 (1978), each of which is incorporated herein byreference). RNAse activity is inhibited by treating all glassware andsolutions with diethylpyrocarbonate.

Following transfer, the filters are dried in a vacuum oven at 80° C.,prehybridized and hybridized in a cocktail containing 5× Denhardt'ssolution, 100 mM sodium phosphate (pH 7.5), 0.5% SDS, 1 mM sodiumpyrophosphate, 100 μM ATP and 50% formamide (see Sambrook et al., supra,1989). Prehybridization and hybridization are performed at 42° C.Following hybridization, the filters are washed twice for 5 min each atroom temperature with 2×SSC/0.5% SDS, then twice for 1 hr each at 65° C.with 0.1×SSC/0.1% SDS (1×SSC=0.15 M NaCl, 0.015 M sodium acetate) andexposed to Kodak XAR-5 Film. Endogenous and antisense TGFβ transcriptsare detected using riboprobes, which can hybridize to the RNA on thefilters.

Sense and antisense riboprobes are prepared by isolating the 1540 bpTGFβ2 cDNA insert in pSTGFB2 and subcloning the insert into pGEM-5ZF(Promega; Madison Wis.). The sense or antisense strands are producedfrom the appropriate RNA polymerase promotor, SP6 or T7, as described bythe manufacturer. These full length RNA probes are used to detect senseand antisense TGFβ nucleic acid sequences. Alternatively, uniquerestriction sites within the TGFβ2 cDNA can be used to linearize pSTGFB2and shorter, more specific sense or antisense RNA probes can besynthesized.

For DNA analysis, 20 μg aliquots of genomic DNA isolated from recoveredcells are digested with various restriction enzymes and sizefractionated on a 1% agarose slab gel in 40 mM Tris-acetate/2 mM EDTA.Following electrophoresis, the DNA transferred to nylon membrane using10×SSC (Sambrook et al, supra, 1989). The blots are hybridized withspecific labeled probes as described above to detect the transgene ofinterest.

B. Murine Ovarian Teratoma Cells

The results described above indicate that inhibition of TGFβ expressionin 9L gliosarcoma cells can enhance a host's systemic immune response tothe tumor cells, regardless of whether an immunostimulatory agent suchas IL-2 is present during immunization. However, different results wereobserved when murine ovarian teratoma cells were examined.

Murine ovarian teratoma (MOT) cells, which produce high levels of TGFβ,cannot easily be crown in tissue culture and, therefore, cannot begenetically modified using a retroviral vector in vitro. Fresh MOT cellswere isolated from murine as cites and were transfected withpCEP4/TGGβ2, which expresses antisense TGFβ2, by electroporation.Expression of TGFβ in MOT cells and in MOT cells that were geneticallymodified to express antisense TGFβ2 was assayed using the mink lungepithelial cell assay. Endogenous TGFβ transcripts are monitored bynorthern blot analyses utilizing sense and antisense specificriboprobes, as described above.

Packaged retroviral vector LNCX/IL2 was used to transduce allogeneicfibroblasts obtained from BALB/c mice to produce CE cells. Severaltransduced clones were selected and expression of IL-2 was determinedusing a commercially available kit (T-Cell Diagnostics, Cambridge,Mass.). The antisense TGFβ2 modified MOT cells were mixed with CE cellsand were injected into C3 H mice. In control experiments, unmodified MOTcells, alone, or in combination with CE cells, or MOT cells geneticallymodified to express antisense TGFβ2, alone, were injected into C3H mice.Fourteen days later, 1×10⁴ live MOT cells were infectedintraperitoneally into the mice and tumor growth was monitored.

In contrast to the results observed for 9L gliosarcoma cells,immunization only with MOT cells hat were genetically modified toexpress antisense TGFβ2 did not significantly increase the survival ofthe animals (FIG. 2). All of the unimmunized mice and the mice receivingMOT cells expressing antisense TGFβ2 were dead within four weeks.Immunization with unmodified MOT cells or with a combination of MOTcells and CE cells resulted in survival of one or two mice,respectively. However, these results were not significantly differentfrom results obtained for the unimmunized mice. In contrast,immunization with a combination of CE cells and MOT cells expressingantisense TGFβ resulted in survival of five of seven mice after 42 days(FIG. 2).

The results of the experiments described herein indicate that, in somecases, immunization with a tumor cells that are genetically modified toprevent the expression of an immunosuppressive agent such as tumor cellsmodified to express an antisense TGFβ2 combined with animmunostimulatory agent such as IL-2 can significantly increase thesurvival of a host to a cancer. In particular, the results indicate thatthere s no detrimental effect of using the combined treatment, whichprevents or inhibits expression of an immunosuppressive agent andprovides an immunostimulatory agent.

Although the invention has been described with reference to the aboveexamples, it is understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

We claim:
 1. A method of stimulating an immune response against acancer, comprising administering to a subject a therapeuticallyeffective amount of genetically modified tumor cells containing agenetic construct expressing a TGFβ inhibitor effective to reduce orinhibit the expression of TGFβ, wherein said genetically modified tumorcells are of the same tumor type obtained from said subject or are donortumor cells, which are of the same histologic type as the subject'stumor cells.
 2. The method of claim 1, wherein said tumor cells arecarcinoma cells, glioma cells, sarcoma cells, lymphoma cells, melanomacells or leukemia cells.
 3. The method of claim 2, wherein said tumorcells are carcinoma cells.
 4. The method of claim 2, wherein said tumorcells are glioma cells.
 5. The method of claim 1, wherein saidimmunosuppressive agent is TGFβ2.
 6. The method of claim 2, wherein saidtumor cells are sarcoma cells.
 7. The method of claim 1, wherein saidgenetic construct expressing a TGFβ inhibitor comprises an antisensenucleic acid sequence.
 8. The method of claim 2, wherein said tumorcells are lymphoma cells.
 9. The method of claim 2, wherein said tumorcells are melanoma cells.
 10. The method of claim 1, wherein said tumorcells comprise carcinoma cells.
 11. The method of claim 2, wherein saidtumor cells are leukemia cells.
 12. A composition for stimulating animmune response against a cancer, comprising tumor cells that aregenetically modified to contain a genetic construct expressing a TGFβinhibitor, wherein said genetically modified tumor cells are of the sametype obtained from said subject or are donor tumor cells, which are ofthe same type as the subject's tumor cells.
 13. The composition of claim12, wherein said tumor cells are carcinoma cells, glioma cells, sarcomacells, lymphoma cells, melanoma cells or leukemia cells.
 14. Thecomposition of claim 13, wherein said tumor cells are carcinoma cells.15. The composition of claim 13, wherein said tumor cells are gliomacells.
 16. The composition of claim 12, wherein said immunosuppressiveagent is TGFβ2.
 17. The composition of claim 13, wherein said tumorcells are sarcoma cells.
 18. The composition of claim 12, wherein saidconstruct expressing a TGFβ inhibitor comprises an antisense nucleicacid sequence.
 19. The composition of claim 13, wherein said tumor cellsare lymphoma cells.
 20. The composition of claim 13, wherein said tumorcells are melanoma cells.
 21. The composition of claim 12, wherein saidtumor cells comprise carcinoma cells.
 22. The composition of claim 13,wherein said tumor cells are leukemia cells.